Multiple sclerosis (hereinafter abbreviated as MS) is an inflammatory demyelinating disease of the human central nervous system with heterogeneous pathophysiological and clinical manifestations and a very complicated etiology (Hafler et al., 2004. J. Clin. Invest. 113: 788-794; Kornek et al., 2003. Brain Res. Bull. 61: 321-326). Progression of the disease in humans leads to destruction of the myelin sheath and that ultimately affects the ability of nerves to conduct electrical impulses (Schwartz, R. S., 1993, in Fundamental Immunology, ed. Paul, W. E. (Raven, New York), pp. 1033-1097).
The viral mimicry hypothesis was formulated to explain the initiation of this pathology (Merkler et al., 2006. J. Clin. Invest. 116: 1254-1263). At present, however, the true triggering mechanisms of the disease have not been clearly identified (Hohlfeld et al., 2004. Proc. Natl. Acad. Sci. USA 101 (Suppl. 2): 14599-14606).
It has been demonstrated that some of the proliferating T-cells in MS patients are directed towards MBP (Allegretta et al., Science, 247, 718-721, 1990) and that human T-cells can recognize multiple epitopes on the MBP molecule (Richert et al., J. Neuroimmun 23, 55-66, 1989). MBP also appears to be capable of activating some T-cells without the involvement of antigen presenting cells (Altman et al., Eur. J. Immun. 17, 1635-1640, 1987).
Despite strong and commonly accepted evidence for immune T cell involvement in development and progression of MS in humans and experimental animal models of the disease, the contributions of a specific B cell response to myelin sheath destruction is much less investigated (Klawiter et al., 2007. Curr. Neurol. Neurosci. Rep. 7: 231-238; Nikbin et al., 2007. Int. Rev. Neurobiol. 79: 13-42).
Ample data indicate that a significant portion of MS cases is characterized by the presence in the blood of auto antibodies directed against MBP components (Reindl et al., 1999, Brain, 122, 2047-2056; Genain et al., 1999, Nat. Med. 5, 170-175). Moreover, high resolution microscopic analysis detected myelin-specific auto antibodies in the regions of demyelination plaques in human MS and a MS-like disease of marmosets, suggesting their direct contribution to myelin destruction (Genain et al., 1999, Nat. Med. 5, 170-175). Although the mechanism of the autoantibody role in MS pathogenesis is unknown, auto antibodies to MBP and myelin oligodendrocyte glycoprotein (MOG) were proposed as biomarkers for clinical prognosis of MS (Berger et al., 2003, N. Engl. J. Med. 349, 139-145.10). Similar immunoglobulins were also found in mice with induced experimental allergic encephalomyelitis (EAE), which is an animal model of MS (Fritz et al., 1983, J. Immunol. 130, 191-194). Increased titers of auto antibodies to MBP were observed in the cerebrospinal fluid (CSF) of patients with active forms of MS (Warren at al., Ann Neurol 209:20-25, 1986). Clinically, MS is characterized by phases of disease activity such as acute relapses or chronic progression, and by phases of clinical remission. Active MS is associated with increased levels of intrathecally appearing auto antibodies to MBP (Warren et al., Ann Neurol 209:20-25, 1986; Catz et al., Can J Neurol Sci 13:21-24, 1986). These antibodies are found predominantly in free (F) form during acute relapses and predominantly in bound (B) form when the disease is insidiously progressive (Warren et al., Ann Neurol 209:20-25, 1986). The therapy of relapsing-remitting multiple sclerosis (RRMS) patients with rituximab a monoclonal antibody, which selectively targets and depletes CD20+ B lymphocytes, appears safe and effective to some extend (Stüve, O. et al., Long-term B-lymphocyte depletion with rituximab in patients with relapsing-remitting multiple sclerosis, Arch Neurol. 2009 February; 66(2):259-61).
Accordingly to that existing scientific background, the approach to treat MS using the immunomodulation with the whole MBP molecule and numerous peptides representing fragments and homologs of MBP protein and its fragments has been developed during last decade. As it is well known human MBP consists of 170 amino acid residues. In scientific and patent literature a fragment of MBP is labeled according to positions of its first and last amino acid residues counted from the C-terminus of full MBP sequence (SEQ ID NO: 1).
In WO 9612737 a number of peptides (human MBP fragments) having “T-cell activity” (i.e. the ability to influence on activity or functions of immune T-cells or their subpopulations) were provided for preventing and treating of MS and has been disclosed as suitable for therapeutic use. To reveal the T-cell recognized epitopes in MBP, 16 short (each about 20 amino acid residues in length) and three more long (82-100, 83-105, 141-165) “overlapping” each other peptide sequences have been tested on their ability to stimulate of in vitro proliferation of peripheral blood cells of MS patients. The amino acid structure of peptides provided by inventors corresponds to the following MBP fragments: 11-30; 11-29; 11-31; 83-105; 82-105; 82-104; 80-98; 82-102; 80-104; 80-102; 111-130; 111-129; 141-165; 101-125. Also, these authors disclosure combination of the inventive peptides with others peptides known from prior art as having T-cell activity of MBP: 13-25; 31-50; 61-80; 82-92; 82-96; 82-97; 82-98; 82-100; 82-100; 83-100; 83-101; 84-97; 84-100; 85-100; 86-105; 87-99; 87-99 [91K>A]; 88-100; 88-99; 82-100; 111-135; 122-140; 139-170; 141-160; 142-166; 142-168; 146-160; 153-170.
Wraith D. C. and co-authors provide a method for selecting of tolerogenic peptides capable of binding to an MHC class I or II molecules without further antigen processing and using these peptides in a pharmaceutical composition for treatment and/or prevention of multiple sclerosis. A number of peptides corresponded to fragments 1-24; 15-39; 30-54; 45-69; 60-84; 75-99; 90-114; 105-129; 120-144; 135-159; 150-170; 131-145; 132-146; 133-147; 134-148; 135-149; 136-150; 137-151; 138-152; 139-153; 140-154; 141-155; 142-156; 143-157; 144-158 of human MBP have been synthesized and used by these authors for identification of human MBP epitopes recognized by MS-patients' peripheral blood cells and then for study of ability of these peptides to bind to MHC class I or class II molecules. A number of short peptides, active in respect of modulation of immune T-cell functions were selected and claimed for MS treatment: 134-148; 135-149; 136-150; 137-151; 138-152; 139-153; 140-154; 30-44; 80-94; 83-99; 81-95; 82-96; 83-97; 84-98; 110-124; 130-144; 131-145; 132-146; 133-147 (see Applications: EP1918298, U.S. Ser. No. 11/979,224, WO 03/64464).
In US2005209156 a peptide selected from MBP fragment (75-98) having the amino acid sequence of AGAPVVHPPLAIVTPAT (SEQ ID NO: 5) including substitutions, additions or deletions thereof, has been disclosed for MS treatment.
In U.S. Pat. No. 5,858,980 the MBP fragments 84-102 and 143-168 were identified as containing immunodominant T-cell recognized epitopes of MBP active in the development of MS, and peptide comprising the amino acid sequence of ENPVVHFFKNIVTPRT (SEQ ID NO: 6) (MBP fragment 83-98) and their analogs and AQGTLSKIFKLGGRD (SEQ ID NO: 7) (MBP fragment 146-160) as well as a pharmaceutical composition comprising the peptides were provided.
Warren K. G. with coauthors provides soluble synthetic peptides, useful to neutralize anti MBP auto antibodies, having the amino acid sequence correspond to following amino acid residues of human MBP: 61-75; 64-78; 69-83; 75-95; 69-83; 80-97; 91-106; 84-93; 85-94; 86-95; 87-96; 82-98. These peptides overlap MBP sequence from 61 to 106 of its amino acid residues (see WO 98/45327). One of the selected peptides (namely MBP 75-95) showed its ability to bound free antibodies to MBP both in vitro and in vivo (in blood of MS-patients). On the contrary, non-binding, control peptide MBP 35-58 had no effect on free and bound antibodies to MBP in MS patients.
Some of MBP based peptides and DNA vaccines has been evaluated in clinical trials: Bayhill Therapeutics DNA-vaccine BHT-3009 containing the gene for whole MBP sequence; altered peptide ligands GP77116 and NBI-5788 from Neurocrine Biosciences; oral tolerogenic composition Myloral from Autoimmune(R) based on whole MBP sequence. However no significant success was observed from those therapies due to serious adverse events or inability to slow MS progression (Hohlfeld et al., Proc Nat Acad. Sci USA. 2004, Oct. 5; 101 (Suppl. 2): 14599-14606.). Now the abovementioned synthetic peptide with a sequence corresponding to amino acid residues 82-98 of MBP (DENPVVHFFKNIVTPRT (SEQ ID NO: 8)) and coded as MBP8298 is being extensively studied in phase II of clinical trials for treatment of progressive multiple sclerosis. The peptide at dose of 500 mg was administered intravenously every six months. It has been shown that responders on the treatment with MBP8298 are the patients with HLA heliotypes DR2 and/or DR4 only (Warren, K. G. et al. European Journal of Immunology, 2006, vol. 13, No 8, pp. 887-895).
Above-mentioned MBP 84-102 fragment (U.S. Pat. No. 5,858,980) was used as active ingredient for preparation for treatment of secondary progressive MS in phase I of clinical trials. No treatment efficacy were detected according to measurement of Expanded Disability Status Scale, Nine Hole Peg Test, new lesions diagnosed by Magnetic Resonance Imaging (Goodkin, D. E. et al. Neurology 2000, vol. 54, pp. 1414-1420)
Thus the search and selection of novel fragments of MBP, oligopeptides having therapeutic activity against MS represents non-trivial task and medicinal properties of any of such oligopeptides with respect to MS treatment cannot be predicted beforehand by a person skilled in the art even on the basis of extensive preclinical in vitro and/or in vivo testing. The identification and selection of such new effective peptides may be achieved through discovery of novel immunopathological mechanisms involved into development and progression of MS.
Earlier we have presented evidence that a small fraction of anti-MBP auto antibodies circulating in blood of MS patients demonstrates site-specific proteolytic cleavage of the MBP molecule and represents novel immunopathological mechanism of myelin destruction and progression of MS (Ponomarenko et al. Immunology Letters 103, 2006, pp. 45-50)
Accordingly, the object of the present invention is to determine novel oligopeptides which would be able to neutralize epitope-specific anti MBP auto antibodies involved into binding and catalytic degradation of MBP in course of progression of Multiple Sclerosis (ESAMBPCAA-epitope specific anti MBP catalytic auto antibodies), and new effective methods of treating MS using these novel oligopeptides.
This object is achieved by providing novel oligopeptide moieties (oligopeptides, fusion proteins composed of these oligopeptides and fragments of these oligopeptides), a pharmaceutical composition containing the said oligopeptides and fusion proteins, which neutralize ESAMBPCAA. Surprisingly, despite ESAMBPCAA represent only minor fraction of circulating anti MBP auto antibodies in MS, the inventors of the present invention have discovered that oligopeptides having sequences comprising as short as 6 amino acid residues of SEQ ID NO: 2, which neutralize ESAMBPCAA, demonstrate unexpectedly high efficacy in treatment of MS.
Other advantages and aspects of the present invention will become apparent upon reading the following detailed description of the invention.